Ionizing radiation and normal aerobic metabolism both lead to the formation of reactive oxygen species (ROS), which can give rise to a spectrum of DNA modifications including single-nucleobase lesions, strand breaks, and crosslink lesions. The emphasis of the present application is placed on understanding the cellular recognition and repair of the under-explored, radiation-induced intrastrand and interstrand crosslink lesions of DNA. The proposed experiments are organized according to three specific aims. In Aim #1, we will employ our newly developed shuttle vector method to explore how radiation-induced intrastrand crosslink lesions perturb DNA transcription and how they are repaired in human cells. In Aim #2, we will investigate the formation of radiation-induced interstrand crosslink lesions and the role of NEIL1 in repairing the intrastrand and interstrand DNA crosslink lesions induced by ionizing radiation and other agents. In Aim #3, we will employ a quantitative proteomic method to identify cellular proteins that are capable of binding specifically to duplex DNA harboring a radiation-induced crosslink lesion, and we will assess the roles of these newly identified proteins in repairing the crosslink lesions in mammalian cells. The outcome of the proposed research will provide novel insights into the repair of the radiation-induced intrastrand and interstrand crosslink lesions, and it may result in a paradigm shift in our understanding of the repair of this type of DNA lesions by revealing the novel role of DNA glycosylase NEIL1 in repairing these lesions and by discovering new damage recognition and DNA repair proteins acting on radiation-induced crosslink lesions. Additionally, the proposed proteomic experiments may afford the identification of novel proteins involved in DNA damage response signaling. Therefore, the proposed research will improve significantly our understanding of the adverse human health effects emanating from exposure to exogenous ionizing radiation and it may ultimately lead to the development of enhanced cancer radiotherapy.